Molecular identification of the morphologically cryptic Asian common treefrogs (Anura: Rhacophoridae, Polypedates leucomystax complex) in Thailand

Abstract

New amphibian species in Southeast Asia have been continuously reported. However, the diversity of amphibians in this region is underestimated due to the prevalence of cryptic species. Recent molecular studies revealed that many amphibian species in Southeast Asia including Polypedates leucomystax comprised cryptic species. In this study, we reveal the cryptic diversity within P. leucomystax complex in Thailand. The 16S rRNA gene (588 to 621 bp) was analyzed from 218 specimens in 54 provinces of Thailand. Then, a different region of 16S (376 to 379 bp) was analyzed using 19 selected sequences from the first analyses and six tadpole from GenBank. Based on phylogenetic analyses, the first tree showed P. leucomystax complex was a monophyletic group. Our specimen collection fell within four clades; A-clade (P. leucomystax), B-clade (Polypedates sp.), C-clade (P. megacephalus) and E-clade (P. discantus). The most intraspecific p-distances were less than or equal to 0.03 while the interspecific p-distance were greater than 0.03. Moreover, the second phylogenetic analysis showed that six tadpole sequences were clustered in the clades of P. cf. mutus1, P. braueri and Polypedates sp. We found P. megacephalus and P. leucomystax were the dominant species in Indochinese and Sundaic subregions, respectively. Furthermore, the B-clade could be considered as a new clade because their genetics differred from the others. Our results concluded that there was cryptic diversity within Asian common treefrogs in Thailand. Molecular identification using 16S marker in our study provided reliable results to identify this cryptic species and will be useful for further studies in population genetics and biogeography.